Live cell imaging using fluorescent microscopy technology has allowed investigation of the transcriptional dynamics of the cell nucleus (Tsien et al., “Seeing the Machinery of Live Cells”, Science 280:1954-1955 (1998); Stephens et al., “Light Microscopy Techniques for Live Cell Imaging,” Science 300.82-86 (2003)). RNA molecules in living cells have been successfully visualized using fluorescent labeled RNA microinjection (Mhlanga et al., “tRNA-Linked Molecular Beacons for Imaging mRNAs in the Cytoplasm of Living Cells,” Nucleic Acid Res. 33: 1902-1912 (2005)), fluorescence in situ hybridization (FISH) (Femino et al., “Visualization of Single RNA Transcripts in Situ,” Science 280:585-590 (1998); Dirks et al., “Visualizing RNA Molecules Inside the Nucleus of Living Cells,” Methods 29:51-57 (2003)), or the green fluorescence protein (GFP), or its derivative (YFP), tagged RNA-binding proteins (Andersen et al., “Nucleolar Proteome Dynamics,” Nature 433:77-83 (2005); Bertrand et al., “Localization of ASH1 mRNA Particles in Living Yeast,” Mol. Cell. 2:437-445 (1998)). However, the global dynamics of RNA distribution and transcriptional activity in the cell nucleus, in relation to the higher order structural organization of DNA, and the temporal and spatial processing and transportation of RNA molecules remains to be analyzed. To achieve this goal, a cell-permeate, RNA-selective fluorescent probe for staining live cells would be essential.
Small cell permeate fluorescent organic molecules are widely used as probes to study living cells (Johnson, “Fluorescent Probes for Living Cells,” Histochem. J. 30:123-140 (1998)). Although cell microinjection (Mhlanga et al., “tRNA-Linked Molecular Beacons for Imaging mRNAs in the Cytoplasm of Living Cells,” Nucleic Acid Res. 33: 1902-1912 (2005)), GFP plasmid transfection (Zhang et al., “Creating New Fluorescent Probes for Cell Biology,” Nat. Rev. Mol. Cell. Bio. 3:906-918 (2002)), and other methods (McNeil et al., “Glass Beads Load Macromolecules into Living Cells,” J. Cell. Sci. 88:669-678 (1987)) to introduce probes into cells have their own advantages for specific study, small molecule imaging probes are often the most practical tool for biological imaging research and medical diagnosis. Many fluorescent dyes are commercially available and stain a variety of living cell organelles, such as the nucleus (Martin et al., “DNA Labeling in Living Cells,” Cytometry Part A 67A:45-52 (2005); Krishan et al., “DAPI Fluorescence in Nuclei Isolated From Tumors,” J. Histochem. Cytochem. 53:1033-1036 (2005)), mitochondria (Pendergrass et al., “Efficacy of MitoTracker Green and CMXrosamine to Measure Changes in Mitochondrial Membrane Potentials in Living Cells and Tissues,” Cytometry Part A 61:162-169 (2004)), lysosomes (Rustom et al., “Nanotubular Highways for Intercellular Organelle Transport,” Science 303:1007-1010 (2004)), and endoplasmic reticulum (Mironov et al., “[Ca2+]i Signaling Between Mitochondria and Endoplasmic Reticulum in Neurons is Regulated by Microtubules. From Mitochondrial Permeability Transition Pore to Ca2+-induced Ca2+ Release,” J. Biol. Chem. 280:715-721 (2005)). In contrast, RNA-specific dyes for staining live cells are not readily available. SYTO®RNASelect (Invitrogen-Molecular Probes, Carlsbad, Calif.) is the only commercially available dye for live cell RNA-imaging (Haulgland, “The Handbook, A Guide to Fluorescent Probes and Labeling Technologies,” tenth ed. M. T. Z. Spence, eds. pp 327, 710-711), but its usefulness has not been widely proven and its molecular structure has not yet been published.
In the past, it has been shown that most nuclear RNA is localized to the nucleolus—a region of the nucleus that is clearly visible using phase contrast microscopy as the densest, phase-dark region of the nucleus. The nucleolus is the key site in the nucleus for synthesis and assembly of ribosomal RNAs (rRNA). Its functions are tightly related to cell growth and proliferation (Lam et al., “The Nucleolus,” J. Cell Sci. 118:1335-1337 (2005); Olson et al., “Conventional and Non-Conventional Roles of the Nucleolus,” Int. Rev. Cytol. 219:199-266 (2002); Carmo-Fonseca et al., “To Be or Not To Be in the Nucleolus,” Nat. Cell Bio. 2:E107-E112 (2000)). It is known that nucleolar rRNA assembly takes place during late telophase and throughout interphase, and that rRNAs disassemble when cells enter mitosis (Hernandez-Verdun et al., “Emerging Concepts of Nucleolar Assembly,” J. Cell Sci. 115:2265-2270 (2002)). However, the detail of nucleolar dynamic mechanisms of intracellular distribution, trafficking and localization throughout a complete cell cycle are not known. Since it is hard to study the nucleolar dynamics using pre-fixed cells, a fluorescent, RNA-selective live cell imaging dye would be greatly advantageous in terms of observing changes in RNA content and distribution, in relation to the organization of DNA within the cell nucleus.
Previously, it has been demonstrated that styryl compounds have the potential to be good live cell fluorescent probes (Rosania et al., “Combinatorial Approach to Organelle-Targeted Fluorescent Library Based on the Styryl Scaffold,” J. Am. Chem. Soc. 125:1130-1131 (2003)), particularly because they have high affinity to DNA (Lee et al., “Development of Novel Cell-Permeable DNA Sensitive Dyes Using Combinatorial Synthesis and Cell Based Screening,” Chem. Comm. 15:852-1853 (2003)). However, finding RNA-selective compounds for live cell imaging is difficult, because small nucleic acid binding molecules generally have better affinity to double-stranded DNA compared to single-stranded RNA. In addition, the living cell system is complicated by the fact that it is rich in proteins and membranes that may lead to non-specific binding of hydrophobic imaging probes. For imaging applications, the RNA binding compound needs to have high cell plasma and nuclear membrane permeability, be well-tolerated by living cells, and resist photobleaching. The present invention is directed to overcoming these and other deficiencies in the art.